The invention relates to antitumor antibiotics. More particularly, the invention relates to analogs of CC-1065 and the duocarmycins having DNA alkylation and antitumor antibiotic activities.
(+)-CC-1065 (1) and the duocarmycins 2 and 3, illustrated in FIG. 1, are natural products having antitumor antibiotic activity through the alkylation of DNA. (Hanka, L. J., et al. J. Antibiot. 1978, 31, 1211; Yasuzawa, T., et al., Chem. Pharm. Bull. 1995, 43, 378; and Takahashi, I., et al., J. Antibiot. 1991, 44, 1045.) Prior studies have shown that the natural products can withstand and may benefit from significant structural modifications to the alkylation subunit and that the resulting agents retain their ability to participate in the characteristic sequence-selective DNA alkylation reaction. (Boger, D. L., et al., Chem. Rev. 1997, 97, 787.) These structural modifications, and the definition of their effects have served to advance the understanding of the origin of the catalysis of the DNA alklation reaction by 1-3. (Harper, D. E. J. Am. Chem. Soc. 1994, 116, 7573; and Warpehoski, M. A., et al., J. Am. Chem. Soc. 1995, 117, 2951.)
These structural modifications have also served to advance the understanding of the origin of the DNA sequence selectivity of 1-3. (Warpehoski, M. A. In Advances in DNA Sequence Specific Agents; Hurley, L. H., Ed.; JAI: Greenwich, Conn., 1992; Vol. 1, p 217; Hurley, L. H. and Draves, P. In Molecular Aspects of Anticancer Drug-DNA Interactions; Neidle, S. and Waring, M., Eds.; CRC: Ann Arbor, 1993; Vol. 1, p 89; and Aristof P. A In Advance in Medicinal Chemistry, JAI: Greenwich, Conn., 1993; Vol. 2, p 67). Two models have been proposed to explain the mechanism of the DNA sequence selectivity of 1-3. One model proposed by Boger states that the DNA sequence selectivity of 1-3 is determined by the AT-rich noncovalent binding selectivity of these agents and their steric accessibility to the adenine N3 alkylation site. (Boger, D. L., et al., Angew. Chem., Int. Ed. Engl. 1996, 3.5, 1439; and Boger, D. L., et al., Biorg. Med Chem. 1997, 5, 263.) This noncovalent binding model accommodates and explains the reverse and offset 5 or 3.5 base-pair AT-rich adenine N3 alkylation selectivities of the natural and unnatural enantiomers of 1 (Boger, D. L., et al., J. Am. Chem. Soc. 1990, 112, 4623; and Boger, D. L., et al, Bioorg. Med Chem. 1994, 2, 115) and the natural and unnatural enantiomers of 2-3. (Boger, D. L., et al., J. Am. Chem. Soc. 1993, 115, 9872; and Boger, D. L., et al., J. Am. Chem. Soc. 1994, 116, 1635.) This noncovalent binding model also requires that simple derivatives of the alkylation subunits exhibit alkylation selectivities distinct from the natural products. It also offers an explanation for the identical alkylation selectivities of both enantiomers of such simple derivatives (5xe2x80x2-AA greater than 5xe2x80x2-TA), and the more extended AT-rich selectivity of the advanced analogs of 1-3 corresponds nicely to the length of the agent and the size of the required binding region surrounding the alkylation site. This model is further supported by the demonstrated AT-rich noncovalent binding of these agents. (Boger, D. L., et al., Chem.-Biol. Interactions 1990, 73, 29; and Boger, D. L., et al., J. Org. Chem. 1992, 57, 1277.) The model is also supported by the correspondance between the observed preferential noncovalent binding and the observed DNA alkylation of these agents. (Boger, D. L, et al., Bioorg. Med. Chem. 1996, 4, 859.) Also the observation that the characteristic DNA alkylation selectivity of these agents does not require the presence of the C-4 carbonyl or even the activated cyclopropane provides further support for the model (Boger, D. L. et al., J. Am. Chem. Soc. 1991, 113, 3980.; and Boger, D. L., et al. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 1431.) The accuracy of this model is further demonstration of the complete switch in the inherent enantiomeric DNA alkylation selectivity that accompanied the reversal of the orientation of the DNA binding subunits with reversed versus extended analogs of the duocarmycins. (Boger, D. L., et al., J. Am. Chem. Soc. 1997, 119, 4977; Boger, D. L., et al., J. Am. Chem. Soc. 1997, 119, 4987; and Boger, D. L., et al., J. Am. Chem. Soc. 1995, 117, 1443.)
The above AT-rich noncovalent binding model contrasts with an alternative proposal in which a sequence-dependent backbone phosphate protonation of the C-4 carbonyl activates the agent for DNA alkylation and controls the sequence selectivity. (Hurley, L. H. J. Am. Chem. Soc. 1995, 117, 2371.)
Structural studies of DNA-agent adducts, 17-19 the C-4 carbonyl of the natural products projects out of the minor groove lying on the outer face of the complexes potentially accessible to the phosphate backbone. (Lin, C. H., et al., J. Mol. Biol. 1995, 248, 162.; and Smith, J. A., et al., J. Mol. Biol. 1997, 272, 237.) However, the relative importance of the C-4 carbonyl positioning to the properties of these agents has not be determined.
What is needed is a series of analogs of (+)-CC-1065 (1) and the duocarmycins 2 and 3 which exploit the AT-rich noncovalent binding model and which retain their DNA binding and alkylating activity and selectivity. What is needed is series of analogs of (+)-CC-1065 (1) and the duocarmycins 2 and 3 which incorporate of iso-CI and iso-CBI (6 and 7). Iso-CI and iso-CBI (6 and 7) are analogs of the CI and CBI alkylation subunits 4 and 5 wherein the key C-4 carbonyl is isomerically relocated to the C-6 or C-8 positions, now ortho to the cyclopropane, as illustrated in FIG. 2. If the AT-rich noncovalent binding model is correct, the relocated carbonyls of iso-CI and iso-CBI (6 and 7) would project into the minor groove inaccessible to the phosphate backbone if participating in an analogous adenine N3 alkylation reaction.
A series of bioactive analogs of (+)-CC-1065 (1) and the duocarmycins 2 and 3 are synthesized. Each of the analogs includes iso-CI or iso-CBI (6 and 7) as a DNA alkylation subunit. The novel DNA alkylation subunits are then conjugated to known DNA binding subunits to form bioactive analogs of (+)-CC-1065 (1) and the duocarmycins 2 and 3. Preferred DNA binding subunits are disclosed herein and in U.S. patent application Ser. No. 09/051,264, incorporated herein by reference.
2-(tert-Butyloxycarbonyl)-1,2,9,9a-tetrahydrocyclo-propa[c]benzo[f]-indol-8-one (31, N-BOC-iso-CBI) and 1-(tert-butyloxycarbonyl)-4-hydroxy-3-[[(methanesulfonyl)oxy]methyl]-2,3-dihydroindole (19, seco-N-BOC-iso-CI) serve as preconjugate forms to the DNA alkylating subunits, i.e., iso-CI or iso-CBI (6 and 7). The approach for synthesizing compounds 31 and 19 was based on a directed ortho metallation of an appropriately functionalized benzene (13) or naphthalene at (24) precursor to regziospecifically install iodine at the C-2 position. Conversion of these respective intermediates to the dihydroindole skeleton utilized an established 5-exo-trig aryl radical cyclization onto an unactivated alkene with subsequent TEMPO trap or the more recent 5-exo-trig aryl radical cyclization onto a vinyl chloride for direct synthesis of the immediate precursors. Closure of the activated cyclopropane to complete the iso-CBI nucleus was accomplished by a selective ortho spirocyclization.
Resolution and synthesis of a fill set of natural product analogs and subsequent evaluation of their DNA alkylation properties revealed that the iso-CBI analogs react at comparable rates and retain the identical and characteristic sequence selectivity of CC-1065 and the duocarmycins. This observation is inconsistent with the prior art proposal that a sequence-dependent C-4 carbonyl protonation by strategically located DNA backbone phosphates controls the DNA alkylation selectivity but is consistent with the proposal that it is determined by the AT-rich noncovalent binding selectivity of the agents and the steric accessibility of the N3 alklation site.
Solvolysis studies indicate that the iso-CBI-based agents have a stability comparable to that of CC-1065 and duocarmycin A and a greater reactivity than duocarmycin SA (6-7xc3x97). Solvolysis studies indicate also indicate that the iso-CBI-based agents are more reactive than the corresponding CBI-based agents (5xc3x97).
Confirmation that the DNA alkylation reaction is derived from adenine N3 addition to the least substituted carbon of the activated cyclopropane and its quantitation (95%) was established by isolation and characterization of the depurination adenine N3 adduct. Consistent with past studies and in spite of the deep-seated structural change in the alkylation subunit, the agents were found to exhibit potent cytotoxic activity that correlates with their inherent reactivity.
One aspect of the invention is directed to DNA alkylating compounds having a DNA alkylating subunit covalently linked to a DNA binding subunit. The DNA alkylating compound is represented by the following structure: 
A preferred DNA binding subunit is a radical represented by the following structure: 
In the above structure, A is selected from the group consisting of NH and O. B is selected from the group consisting of C and N. R2 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl(C1-C6), N-alkyl(C1-C6)3 and a first N-substituted pyrrolidine ring. R3 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl(C1-C6), N-alkyl(C1-C6)3, the first N-substituted pyrrolidine ring. R4 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl, (C1-C6), and N-alkyl(C1-C6)3. R5 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl(C1-C6), and N-alkyl(C1-C6)3 V1 represents a first vinylene group between R2 and R3. However, there are various provisos. If R2 participates in the first N-substituted pyrrolidine ring, then R3 also participates in the first N-substituted pyrrolidine ring. If R3 participates in the first N-substituted pyrrolidine ring, then R2 also particlates in the first N-substituted pyrrolidine ring. If R2 and R3 participate in the first N-substituted pyrrolidine ring, then R4 and R5 are hydrogen. If R2 is hydrogen, then R4 and R5 are hydrogen and R3 is N-alkyl (C1-C6)3. The first N-substituted pyrrolidine ring is fused to the first vinylene group between R2 and R3 and is represented by the following structure: 
In the above structure V1 represents the first vinylene group between R2 and R3. R6 is selected from the group consisting of xe2x80x94CH2CH3(alkyl), xe2x80x94NHCH3(xe2x80x94N-alkyl), xe2x80x94OCH3(O-alkyl), xe2x80x94NH2, xe2x80x94NHNH2, xe2x80x94NHNHCO2txe2x80x2Bu, and a radical represented by the following structure: 
In the above structure, C is selected from the group consisting of NH and O. D is selected from the group consisting of C and N. R7 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl(C1-C6), N-alkyl(C1-C6)3, and a second N-substituted pyrrolidine ring. R8 is selected from the group consisting of hydrogen, hydroxyl, O-alkyl(C1-C6), N-alkyl(C1-C6)3, the second N-substituted pyrrolidine ring. V2 represents the second vinylene group between R7 and R8. However, the following provisos pertain. If R7 participates in the N-substituted pyrrolidine ring, then R8 also particlates in the N-substituted pyrrolidine ring. If R8 participates in the N-substituted pyrrolidine ring only if R7 also particlates in the N-substituted pyrrolidine ring. The second N-substituted pyrrolidine ring is fused to the second vinylene group between R7 and R8 and is represented by the following structure: 
In the above structure, V2 represents the second vinylene group between R7 and R8. R9 is selected from the group consisting of xe2x80x94CH2CH3(alkyl), xe2x80x94NHCH3(xe2x80x94N-alkyl), xe2x80x94OCH3(O-alkyl), xe2x80x94NH2, xe2x80x94NHNH2, and xe2x80x94NHNHCO2txe2x80x2Bu.
Preferred examples include DNA alkylating compounds represented by the following structures: 
Another aspeact of the invention is directed to DNA alkylating compounds represented by the following structure: 
In the above structure, R13 is selected from the group consisting of xe2x80x94C1-C6 alkyl, xe2x80x94NHCH3(xe2x80x94N-alkyl), xe2x80x94OCH3(O-alkyl), xe2x80x94NH, xe2x80x94NHNH2, xe2x80x94NHNHCO2txe2x80x2Bu, and a radical represented by the following structure: 
A preferred embodiment of this aspeact of the invention is represented by the following structure: 
Further aspeacts of the invention are directed to chemical intermediate represented by the following structures: 
Another aspeact of the invention is directed to DNA alkylating compounds having a DNA alkylating subunit covalently linked to a DNA binding subunit covalently linked said DNA alkylating subunit, wherein the DNA alkylating compound being represented by the following structure: 
Preferred DNA binding subunit are as described above for the iso-CBI compounds. Preferred examples of this aspect of the invention include DNA alkylating compounds represented by the following structures: 
Another aspect of the invention is directed to DNA alkylating compounds represented by the following structure: 
In the above structure R1 is selected from the group consisting of xe2x80x94C1-C6 alkyl, xe2x80x94NHCH3 (xe2x80x94N-alkyl), xe2x80x94OCH3(O-alkyl), xe2x80x94NH2, xe2x80x94NHNH2, xe2x80x94NHNHCO2tBu, and a radical represented by the following structure: 
An example of this preferred embodiment is represented by the following structure: 
An other aspect of the invention is directed to chemical intermediates presented by the following structures: 